A Sabin 3-derived poliovirus recombinant contained a sequence homologous with indigenous human enterovirus species C in the viral polymerase coding region

Abstract

Outbreaks of poliomyelitis caused by circulating vaccine-derived polioviruses (cVDPVs) have been reported in areas where indigenous wild polioviruses (PVs) were eliminated by vaccination. Most of these cVDPVs contained unidentified sequences in the nonstructural protein coding region which were considered to be derived from human enterovirus species C (HEV-C) by recombination. In this study, we report isolation of a Sabin 3-derived PV recombinant (Cambodia-02) from an acute flaccid paralysis (AFP) case in Cambodia in 2002. We attempted to identify the putative recombination counterpart of Cambodia-02 by sequence analysis of nonpolio enterovirus isolates from AFP cases in Cambodia from 1999 to 2003. Based on the previously estimated evolution rates of PVs, the recombination event resulting in Cambodia-02 was estimated to have occurred within 6 months after the administration of oral PV vaccine (99.3% nucleotide identity in VP1 region). The 2BC and the 3D(pol) coding regions of Cambodia-02 were grouped into the genetic cluster of indigenous coxsackie A virus type 17 (CAV17) (the highest [87.1%] nucleotide identity) and the cluster of indigenous CAV13-CAV18 (the highest [94.9%] nucleotide identity) by the phylogenic analysis of the HEV-C isolates in 2002, respectively. CAV13-CAV18 and CAV17 were the dominant HEV-C serotypes in 2002 but not in 2001 and in 2003. We found a putative recombination between CAV13-CAV18 and CAV17 in the 3CD(pro) coding region of a CAV17 isolate. These results suggested that a part of the 3D(pol) coding region of PV3(Cambodia-02) was derived from a HEV-C strain genetically related to indigenous CAV13-CAV18 strains in 2002 in Cambodia.

FIG. 1.

The genomic sequence of Cambodia-02. (A) Alignment of the genome of Cambodia-02 (accession no. AB205395) with that of Sabin 3 (accession no. X00925). The numbers in each region represent the percentages of nucleotide identity with the Sabin 3 genome. The numbers in parentheses represent the percentages of amino acid identity. The genomic regions that showed more than 90% amino acid identity are colored with light gray, and genomic regions that showed more than 96% nucleotide identity are colored with dark gray. The nucleotide identity in the 5′ part of the genome (including the 5′NTR and the structural protein coding region) and in the nonstructural protein (NS) coding region are also shown. (B) Similarity plot analysis of Cambodia-02 and Sabin strains (Sabin 1, accession no. AY184219; Sabin 2, accession no. AY184220) calculated by SimPlot. The nucleotide sequence of the Cambodia-02 genome was used as the reference. A window size of 200 bp with an increment of 20 bp was used. (C) Alignment of the genome of Cambodia-02 with that of Sabin 3 near the putative recombination junction in the 2A^pro coding region. The part representing unidentified sequence (from nt 3778 to the 3′ end) is colored with light gray. (D) The nucleotide and amino acid differences in the 5′NTR and the capsid proteins of Cambodia-02. Numbers represent the positions of nucleotides in the 5′NTR or of the amino acid residues in each capsid protein.

FIG. 2.

Phylogenic analysis of the 3D^pol coding region. The phylogenic tree was generated from the nucleotide sequences of the 3D^pol coding region of Cambodian HEV-C isolates, prototype HEV-C strains, and Sabin strains. The location of Cambodia-02 is indicated by an arrow. A putative HEV-C recombinant (CAM2069) is colored with light gray. The nomenclature of the isolates indicates the names of the isolates, serotypes, and the GenBank/EMBL/DDBJ accession numbers. Bootstrap values are shown at the branch nodes. Bar, 0.1 substitution per site.

FIG. 3.

Alignment of the genomes of Cambodian HEV-C isolates. The numbers in each region represent the percentages of nucleotide identity, and the numbers in parentheses represent the percentages of amino acid identity. The genomic regions that showed more than 90% amino acid identity are colored with light gray, and the genomic regions that showed more than 92% nucleotide identity are colored with dark gray. (A) Alignment of Cambodia-02 with a CAV13-CAV18 isolate (CAM1900). (B) Alignment of a CAV17 isolate (CAM2069) with CAM1900 (CAV13-CAV18) and CAM2101 (CAV17). (C) Multisequence analysis of HEV-C isolates and Cambodia-02 by similarity plot analysis calculated by SimPlot. CAM1900 was used as the reference. A window size of 200 bp with an increment of 20 bp was used. The locations of 3C^pro and 3D^pol coding regions are shown in the plot. (D) Alignment of a part of the genome of CAM1900 with that of CAM2069 around the putative recombination junction near the 3C^pro coding region. The part representing unidentified sequence is colored with gray. NS; nonstructural protein, ND; not determined.

FIG. 4.

Phylogenic analysis of the 2BC coding region. The phylogenic tree was generated from the nucleotide sequences in the 2BC coding region of Cambodian NPEV isolates, prototype HEV-C strains, and Sabin strains. The location of Cambodia-02 is indicated by an arrow. The nomenclature of the isolates indicates the names of the isolates, serotypes, and the GenBank/EMBL/DDBJ accession numbers. Bootstrap values are shown at the branch nodes. Bar, 0.1 substitution per site.